Modular micro-PCR system for the onsite rapid diagnosis of COVID-19

Effective containment of the COVID-19 pandemic requires rapid and accurate detection of the pathogen. Polymerase chain reaction (PCR) remains the gold standard for COVID-19 confirmation. In this article, we report the performance of a cost-effective modular microfluidic reverse transcription (RT)-PCR and RT-loop mediated isothermal amplification (RT-LAMP) platform, Epidax®, for the point-of-care testing and confirmation of SARS-CoV-2. This platform is versatile and can be reconfigured either for screening using endpoint RT-PCR or RT-LAMP tests or for confirmatory tests using real-time RT-PCR. Epidax® is highly sensitive and detects as little as 1 RNA copy per µL for real-time and endpoint RT-PCR, while using only half of the reagents. We achieved comparable results with those of a commercial platform when detecting SARS-CoV-2 viruses from 81 clinical RNA extracts. Epidax® can also detect SARS-CoV-2 from 44 nasopharyngeal samples without RNA extraction by using a direct RT-PCR assay, which shortens the sample-to-answer time to an hour with minimal user steps. Furthermore, we validated the technology using an RT-LAMP assay on 54 clinical RNA extracts. Overall, our platform provides a sensitive, cost-effective, and accurate diagnostic solution for low-resource settings.


Introduction
This instructions for use must be read carefully prior to use and instructions must be followed accordingly. If there are any deviations from this instructions for use (IFU), reliability of this assay results cannot be guaranteed. RESOLUTE 2.0 is available in 100 reactions (CKFG0002) and 50 reactions (CKFG0003).

2.
Intended Use RESOLUTE 2.0 is a real-time RT-PCR test intended for the qualitative detection of nucleic acid from the SARS-CoV-2 virus in nasopharyngeal swabs specimen from individuals with signs and symptoms of infection who are suspected of COVID-19. The test results from the RESOLUTE 2.0 SARS-CoV-2 detection assay should not be used as the sole basis for patient management decisions.

Summary and Explanation
An outbreak of pneumonia caused by a novel coronavirus (SARS-CoV-2) in Wuhan City, Hubei Province, China was initially reported to WHO on December 31, 2019. Coronavirus Disease 2019 (COVID-19) is a new infectious disease and is caused by the SARS-CoV-2 virus. Symptoms include cough, fever, sore throat, shortness of breath and pneumonia. As of 10th April 2020, WHO has reported about 1,500,000 infections with more than 90,000 deaths.
The RESOLUTE 2.0 COVID-19 Real-Time RT-PCR Tests is a molecular in vitro diagnostic test that detects the SARS-CoV-2 virus and is based on widely used nucleic acid amplification technology. The product contains oligonucleotide primers and fluorophore-labelled probes, control material used in RT-PCR and the enzyme mix required for the in vitro qualitative detection of SARS-CoV-2 RNA in respiratory specimens.

4.
Principles of Procedure RESOLUTE 2.0 is based on real-time reverse transcriptase polymerase chain reaction (RT-PCR) technology, for the qualitative detection of SARS-CoV-2 specific RNA. The RESOLUTE 2.0 SARS-CoV-2 detection assay enables the direct amplification of Coronavirus SARS-CoV-2 RNA from nasopharyngeal swabs (NPS) UTM.
In the RESOLUTE 2.0 SARS-CoV-2 detection assay, forward and reverse primers amplify SARS-CoV-2 viral RNA, and are used with fluorescent probes specific to the viral targets. The RESOLUTE 2.0 SARS-CoV-2 panel consists of three (3) assays, an E gene assay which detects members of the Sarbecovirus subgenus of coronavirus, an N gene assay which detects coronavirus type SARS-CoV-2 specifically and an endogenous housekeeping assay targeting the human RNase P gene (RP). The E gene assay amplifies a target from the virus envelope gene and the N gene assay amplifies a target from the virus nucleocapsid gene. The RP assay is included as a control to detect clinical specimen collection failure, cell lysis failure or RT-PCR failure including inhibition of reaction.   The equipment and consumables that is required but not provided in this kit is listed below. The names of vendors or manufacturers are provided as examples of suitable product sources. Users have to conduct verification whether using product sources provided or other product sources.
• Desktop centrifuge with a rotor for 1.5mL and 2mL reaction tubes • Centrifuge that goes up to 1000 x g with a rotor for microtiter plates if using 96-well reaction plates, microcentrifuge if using PCR tubes or strips • Vortex mixer • Micropipettes (adjustable: 2μl, 10 μl, 100 μl, 200 μl and 1000 μl) • Disposable pipette tips with filters and aerosol barriers • 1.5mL microcentrifuge tubes (DNase/RNase free) • Racks for 1.5mL microcentrifuge tubes • 2x 96-well -20°C cold blocks for holding the RT-PCR assay set-up PCR tubes • Disposable PPE, including powder-free gloves and surgical gowns • Compatible options of RT-PCR platforms are provided in Table 3 below  microcentrifuge tubes, pipette tips) for assay setup and handling of clinical specimens. o Wear a clean lab coat and powder-free disposable gloves (not previously worn) when setting up assays. o Change gloves between samples and whenever contamination is suspected. o Keep reagent and reaction tubes capped or covered as much as possible. o Primers, probes (including aliquots), and enzyme master mix must be thawed and maintained on cold block at all times during preparation and use. o Work surfaces, pipettes, and centrifuges should be cleaned and decontaminated with cleaning products such as 20% bleach, "DNAZap™" or "RNase AWAY®" to minimize risk of nucleic acid contamination. Residual bleach should be removed using 70% ethanol. Turn on UV light to disinfect working surfaces for 30 minutes. • Dispose of unused kit reagents and human specimens according to local, state, and federal regulations.
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Reagent Storage, Handling and Stability
• The RESOLUTE 2.0 kit is shipped in a cold chain environment, on dry ice or coolant (between -25°C and -15°C).
The components of the kit should arrive cold. If the kit components are not cold upon receipt, or if vials have been compromised during shipment, contact your local distributor for assistance. • All components are to be stored between -25°C and -15°C upon arrival.
• Protect from light.
• Before initial use, check the product and its components for: o Integrity o Completeness with respect to number, type and filling o Correct labelling • Use of this product is limited to personnel specially instructed and trained in the techniques of real-time RT-PCR. • Primer-Probes Mixes, control templates and enzyme mixes must be thawed and kept on a cold block at all times during preparation and use. • Avoid microbial and nuclease (DNase/RNase) contamination of the specimens and the components of the Kit.
• Always use DNase/RNase-free disposable pipette tips with aerosol barriers.
• Always wear protective disposable powder-free gloves when handling kit components.
• The workflow in the laboratory should proceed in a unidirectional manner. Use separated and segregated working areas for: i. sample preparation ii. reaction setup and iii. amplification/detection activities • Always wear disposable powder-free gloves in each area and change them before entering a different area.
• Dedicate supplies and equipment to the separate working areas and do not move them from one area to another. • Store positive and/or potentially positive material separately from all other components of the Kit.
• Do not open the reaction tubes/plates post amplification, to avoid contamination with amplicons.
• Do not autoclave reaction tubes after the RT-PCR, since this will not degrade the amplified nucleic acid and risks contaminating the laboratory area. • Do not use expired components, refer to the label for expiration date.

Specimen Collection, Handling and Storage
Acceptable Specimens • Nasopharyngeal swabs in universal transport medium (UTM) or equivalent.
• Types of transport medium validated to be compatible with RESOLUTE 2.0 include the Copan Universal Transport Medium™ (UTM®, Copan Diagnostics), ASAN Transport Medium (ASANPHARM) and the BD universal viral transport (UVT) system (Becton Dickinson). • Compatibility of other types of transport medium must be validated by the users.

Biosafety Precautions on Specimen Handling
• Specimens should always be treated as infectious and/or biohazardous in accordance with safe laboratory procedures. • Users should handle clinical specimen in biosafety level 2+ and above, and observe the safety requirement of a biosafety level 2+ lab. • Discard samples and assay waste according to your local safety regulations.

Specimens Preparation
• Samples to be collected into sterile, labelled tubes and shipped at 2-8°C on frozen gel packs.
• Collection of nasopharyngeal or oropharyngeal swabs: flocked swabs are preferred especially for nasopharyngeal swabs. Sterile dacron or Rayon tipped swabs may also be used for oropharyngeal swabs. Specimens in swabs are to be placed immediately in sterile tubes containing 3mL of sterile UTM. • For delayed testing, specimens can be stored at -70°C and below.

Reagent and Controls Preparation
Important Notes Before Starting: • Plate or tube layout is dependent on the throughput of the laboratory.
• To ensure validity of each run, at least one Positive Control (component 3) and one Negative Control (component 4) must be included. • Once thawed, all reagents should be kept cold either on ice or cool block throughout the preparation and setup. • It is strongly recommended that users prepare and set up the reaction in segregated areas as follows:

Reagent Preparation Room:
a) In a PCR workstation pre-cleaned with 70% ethanol and UV-irradiated, prepare the primary Master Mix. b) Transfer the tubes of primary Master Mix via a transfer box to the Sample Preparation Room.

Sample Preparation Room:
a) Aliquot the completed Master Mix into the respective wells, followed by Negative Control (component 4), clinical nasopharyngeal swab UTM, and finally, Positive Control Template (component 3), into an appropriate optical 96-well reaction plate or an appropriate optical reaction tube according to a predefined layout. b) Seal or cap the loaded optical 96-well reaction plate or optical reaction tube, and centrifuge in a centrifuge with an appropriate rotor for 30 seconds at approximately 1000 x g to bring down the reaction mix to the bottom of the optical 96-well reaction plate or optical reaction tube. c) Transfer the sealed or capped optical 96-well reaction plate or optical reaction tube via a transfer box to the PCR Room.

PCR Room:
a) Load the optical 96-well reaction plate or optical reaction tube in the thermal cycler preloaded with the appropriate cycling profile and start run.

RT-PCR Reaction Master Mix Preparation
• Do not combine components of assay kits with different lot numbers.  Table 4 below. Mix the master mix by gentle pipetting or pulse-vortexing, and centrifuge briefly before use.    o The Startup Wizard screen will appear. Select the following:

Setting-up of a real-time RT-PCR run on a BioRad CFX96™ TOUCH Real-Time
▪ Instrument: CFX96 ▪ Run type: User-defined ▪ After clicking User-defined, the Run Setup screen will appear. ▪ After clicking Create New, the Protocol Editor screen will appear to create a new protocol. • Set up the thermal cycling conditions as detailed in Table 5 below.

Interpretation of RESOLUTE 2.0 test results MUST take into consideration the CT values, as well as the shape of the amplification curve.
Recommendation on the interpretation of test run validity and test result interpretation are as summarized in Table  6 and Table 7.

Limit of Detection (LoD)
LoD studies determines the lowest detectable concentration (in RNA copies/μL) of the SARS-CoV-2 pseudo virus (Armoured RNA Quant SARS CoV2 Panel, Asuragen, Cat no. #52036) at which approximately 95% of all (true positive) replicates test positive.
Known concentration of serially diluted SARS-CoV-2 pseudo virus was spiked into clinically negative nasopharyngeal swab UTM, which have been verified to be SARS-CoV-2 negative by the cobas® SARS-CoV-2 assay (Roche, P/N 09175431190). Analytical LoD was determined by first establishing a preliminary LoD, then by repeating the LoD tests using 20 replicates of two-fold serial dilution of the pseudo virus spiked into the clinical specimen matrix mimic.
RESOLUTE 2.0 test was performed using 5μl of UTM per standard protocol in Table 4 and detected on a BioRad CFX 96 TOUCH Real-Time PCR Instrument.

Limit of Blank (LoB)
LoB studies determine the highest apparent analyte concentration expected to be found when replicates of a blank sample containing no analyte are tested.
To determine the LoB of RESOLUTE 2.0 SARS-CoV-2 detection assay, a 'blank sample' was created using UTM.
19 replicates of this 'blank sample' were tested using the RESOLUTE 2.0 assay per standard protocol in Table 4.
Detection was performed using a BioRad CFX 96 TOUCH Real-time PCR instrument. Table 9 shows the result of the LoB study.

Analytical Specificity
In silico analysis Primers in the RESOLUTE 2.0 E gene and N gene assays were individually mapped to the NCBI sequences of the organisms listed in Table 10. Potential cross reactivity due to PCR amplification was noted if any of the primer pairs mapped to a sequence, with more than 50% homology, on opposite strands less than 1000 nucleotide apart.
No potential unintended cross reactivity is expected based on this in silico analysis.   Clinical Performance The assays were tested with 66 1 nasopharyngeal swab specimens (31 SARS-CoV-2 positives, 2 presumptive positive, 34 SARS-CoV-2 negatives) in universal transport medium (UTM). These were specimens previously tested for presence or absence of SARS-CoV-2 by the cobas® SARS-CoV-2 assays (Roche, P/N 09175431190). Samples were randomized and blinded in the clinical performance evaluation.
RESOLUTE 2.0 test was performed using 5μl of UTM per standard protocol in Table 4 and detected on a BioRad CFX 96 Real-Time PCR Instrument.
All clinical specimens were tested positive for human nucleic acids using the RP assay.
Results of the clinical performance evaluation between RESOLUTE 2.0 SARS-CoV-2 detection Kit and the cobas® SARS-CoV-2 assays are summarized in Table 14.

Interfering Substances
Interference study was conducted to demonstrate that potentially interfering substances that may be found in the upper respiratory tract do not interfere with the detection of SARS-CoV-2 RNA in the RESOLUTE 2.0 assays.
Ten (10) potential interfering substances in the concentration listed below were tested in Table 15. The analytical performance of the RESOLUTE 2.0 assays was assessed in the presence and absence of each of these substances.
None of the substances listed in Table 15 tested in the interference study demonstrated interference. c. with 5% Afrin Nasal Spray, while there was no effect on test performance based on CT values, we noted a reduction in the E gene RFU. The interference study reporting is limited to the percentage Afrin tested in this report.

Limitations
• As a comprehensive interference study was not performed for the RESOLUTE 2.0 SARS-CoV-2 Direct Detection Kit. Samples with abnormally high background signal should be repeated with RNA extraction, followed by conventional RT-PCR test. • All users, analysts, and any person reporting diagnostic results should be trained to perform this procedure by a competent instructor. They should demonstrate their ability to perform the test and interpret the results prior to performing the assay independently. • Performance of the RESOLUTE 2.0 SARS-CoV-2 Detection Assay has only been established in nasopharyngeal swabs specimens. • Negative results do not preclude COVID-19 infection and should not be used as the sole basis for treatment or other patient management decisions. Optimum specimen types and timing for peak viral levels during infections caused by SARS-CoV-2 have not been determined. Collection of multiple specimens (types and time points) from the same patient may be necessary to detect the virus. • A false negative result may occur if a specimen is improperly collected, transported or handled. False negative results may also occur if amplification inhibitors are present in the specimen or if inadequate numbers of organisms are present in the specimen. • Do not use any reagent past the expiration date.
• Test performance can be affected because the epidemiology and clinical spectrum of infection caused by SARS-CoV-2 is not fully known. For example, clinicians and laboratories may not know the optimum types of specimens to collect, and, during the course of infection, when these specimens are most likely to contain levels of viral RNA that can be readily detected. • Detection of viral RNA may not indicate the presence of infectious virus or that SARS-CoV-2 is the causative agent for clinical symptoms. • The performance of this test has not been established for monitoring treatment of SARS-CoV-2 infection.
• The performance of this test has not been established for screening of blood or blood products for the presence of SARS-CoV-2. • This test cannot rule out diseases caused by other bacterial or viral pathogens. • RESOLUTE 2.0 COVID-19 Real-Time RT-PCR Test results shall not be used as the sole means for clinical diagnosis and treatment. • RNA viruses in particular show substantial genetic variability. Although continuous efforts were made to monitor potential mutation in the target regions that might result in mis-match between the primers probes and the target